A method for the identification of promoters recognized by RNA polymerase containing a particular sigma factor: cloning of a developmentally regulated promoter and corresponding gene directed by the Streptomyces aureofaciens sigma factor RpoZ.
We have developed a method for the identification of promoters recognized by a particular sigma factor of RNA polymerase, based on a two-compatible plasmid system in Escherichia coli (Ec). Using the method, a DNA fragment containing the promoter, PREN40, recognized by sporulation-specific Streptomyces aureofaciens (Sa) sigma factor RpoZ, was cloned. High-resolution S1 nuclease mapping using RNA prepared from Ec, and Sa from various developmental stages has shown a high degree of similarity of PREN40 to consensus sequence of flagellar and chemotaxis promoters. The promoter was induced at the time of aerial mycelium formation, and was off in the Sa strain with the rpoZ-disrupted gene. A promoter-bearing DNA fragment was inserted into the promoter-probe plasmid pARC1 to give expression patterns consistent with the results of direct RNA analysis. The region downstream of the promoter was cloned in Sa. Sequence analysis revealed an open reading frame (ORF) of 283 amino acids (Mr 30006), encoding a highly basic (pI 12.35) protein with high percentage of serine, threonine and alanine (41.8%).
Nováková
R
R
Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 842 51, Bratislava, Slovak Republic.
Sevcíková
B
B
Kormanec
J
J
D000409:Alanine / Q000032:analysis; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D012694:Serine / Q000032:analysis; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism*; D013171:Spores, Bacterial / Q000235:genetics / Q000502:physiology; D013304:Streptomyces aureofaciens / Q000235:genetics* / Q000502:physiology; D013912:Threonine / Q000032:analysis; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C087501:RpoZ protein, Streptomyces aureofaciens; D012808:Sigma Factor; D013912:Threonine; D012694:Serine; D012321:DNA-Directed RNA Polymerases; D000409:Alanine